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The application of fatty acid methyl ester analysis (FAME) for the identification of heterotrophic bacteria present in decaying Lede-stone of the St. Bavo Cathedral in Ghent
ELSEVIER
The Science
of the Total
Environment
167 (1995)
241-247
The application of fatty acid methyl ester analysis (FAME) for the identification of heterotrophic bacteria present in decaying Lede-stone of the St. Bavo Cathedral in Ghent P. Descheemaeker*, J. Swings Laboratorium
voor Microbiologic,
Vakgroep
Biochemie, Fysiologie en Microbiologic, 35, B-9000 Ghent, Belgium
Universiteit
Gent, K. L. Ledeganckstraat
Abstract
The heterotrophic bacterial community presentin decaying Lede-sandstoneof the Cathedral of Ghent (Belgium) wasstudied.Two-hundred thirty-two heterotrophic bacterial strainswere isolatedof which 162were studied by fatty acid methyl ester analysis(FAME). One-hundred forty isolateswere Gram-positive; 101strainsbelong to the genus Micrococcus; nine strainswere identified as M. kristinae. One large cluster of 83 strainswasrelated to M. luteus/M. lylae. Other Gram-positive strainswere related to Arthrobacter. Of the 22 Gram-negativestrains,sevenstrainswere related to Pseudomonas uesiculati. A number of Gram-negativeand Gram-positive isolatesformed separateclusters which could not be identified. The possiblerole of the bacterial isolatesin stone degradationwas examinedby a zone-clearing test on a calcite-containingmedium. Of the 152 strains examined,only nine showedclearing zones after 7 days of incubation at 25°C 20 odd strainsshoweda dissolutionof calcite after 14 days, whereasall other strainsshowedno clearing zones,even after prolonged incubation (up to 3 months).The zone-clearingtest showed the samereactionswhen incubated at 24°C or at 19°C. Keywords: Fatty acid methyl ester analysis(FAME); Heterotrophic bacteria; St. Bavo Cathedral, Ghent; Micrococcus; Arthrobacter; Stone decay
1. Introduction Chemoorganotrophic bacterial growth on stone is dependent on the available carbon compounds for an energy and carbon source. Stonework seems to offer enough organic substances for heterotrophic growth (Rosvall, 1986). The organic
*Corresponding
author.
0048-9697/95/$09.50 0 1995 Elsevier SSDI 0048-9697(95)04585-B
Science
BV. All rights
reserved.
compounds may have different origins: (1) phototrophic organisms(cyanobacteria, algae, lichens) absorb sunlight as an energy source and CO, as a carbon source to establish their life cycle. Carbohydrates excreted by these organisms or the organic matter released after cell death can be used as carbon source for chemoorganotrophic growth; (2) a range of hydrocarbons originate from industrial, traffic or domestic burning pollu-
242
P. Descheemaeker, J. Swings
/Science
tants, dirt and dust particles (Warscheid et al., 1991) and (3) sedimentary rocks may contain diagenetic organic material sufficient to support chemoorganotrophic growth (Eckhardt, 198.5). Chemoorganotrophic bacteria are thought to play a role in stone decay (May et al., 19931, although experimental proof of microbial weathering under natural (low nutrient) conditions is still lacking. The mere occurrence of microorganisms on stone surfaces does not automatically imply destructive action, but these microbial populations may be involved in the aesthetic detrimental appearance of historic monuments (Warscheid et al., 1988). The involvement of microbial weathering processes on a specific building can only be assessed by a complete description of the microbial community and proof that this community is mechanistically responsible for deterioration (Palmer, 1992). Studies on the identification of the heterotrophic microbial populations present on decaying stone are rare. Predominant Gram-positive (Micrococcus) and Gram -negative (Flavobactetium, Pseudomonas) bacteria have been identified (Bassi et al., 1986; Lewis et al., 1988). Tayler and May (1991) found during the winter and spring months both Gram-negative (e.g. Flavobacterium / Cytophaga, Actinobacter) and Gram-positive (e.g. Corynebactetiurn, Micro-
coccus) bacteria whereas in the drier summer months, only Gram-positive forms (predominantly Bacillus sp.> were found, suggesting a seasonal variation. It was the objective of this study to identify the heterotrophic microbial population present on deteriorating Lede-stone of the St. Bavo cathedral in Ghent, and to determine their distribution over the different sampling sites. 2. Materials and methods 2.1. Description of the Lede-stone and study site
The deteriorating Lede-stone of the St. Bavo Cathedral of Ghent (Belgium) was sampled. The cathedral is largely built of Balegem (or Lede) stone. The Balegem stone is a light, yellowishgreyish, sandy limestone of Tertiary age. It contains a significant amount of rather fine detrital
of
the Total Environment 167 (1995) 241-247
quartz grains, and some glauconite and calcareous skeletons, cemented by a micro-crystalline calcium carbonate matrix. The total calcite content is 40-60%. It is the typical building material for the Gothic, Renaissance and Baroque monuments of Flanders (Nijs, 1985; Leysen et al., 1990). 2.2. Sample collection and treatment
Stone samples of lo-15 g were peeled off from the surface of severely deteriorated Lede-stones from the northern (five samples), the southern (seven samples) and the western side (two samples) of the cathedral, 1.5-2 m above the ground. Immediately after collection, the stone samples were crushed to a fine powder in an alcoholflamed mortar under a laminar flow hood. One gram of the powder was shaken for 1 h in 10 ml of a sterile water solution containing 0.001% Tween 80 and 0.85% NaCl (Lewis and May, 19851, diluted 100 times and spiral plated in triplicate on Petri dishes containing 30 ml PYGV medium to enumerate heterotrophic bacteria. The PYGV medium contains per litre: 0.025% bacteriological peptone, 0.025% yeast extract, 0.025% glucose, 20 ml Hutners mineral salts (Vreeland, 1991), 0.005% actidione and 1.5% agar. After sterilization, 10 ml of a vitamin solution was added containing per litre MilliQ water: 4 mg biotin, 20 mg pyridoxine hydrochloride, 10 mg thiamine hydrochloride, 10 mg Ca-pantothenate, 10 mg p-aminobenzoic acid, 4 mg folic acid, 10 mg riboflavin, 10 mg nicotinic acid and 0.2 mg vitamin B,z (Palmer et al., 1991). The pH of the stone samples was measured by suspending 1 g of the powdered stone in 15 ml of 1 M KCl. 2.3. Enumeration and isolation of chemoo%anotrophic bacteria
The inoculated PYGV media were incubated for 15 days at 25°C and counted each 5 days. Two-hundred thirty-two heterotrophic bacterial strains were isolated representative of the different dominant colony types. Each strain was purified on PYGV medium. Pure cultures were stored under liquid nitrogen. For each pure culture colony morphology, cell morphology, Gram stain, pigmentation and spore formation was recorded.
P. Descheemaeker,
2.4. Fatty acid methyl ester (FAME)
J. Swings /Science
of the Total Environment
analysis
All pure cultures were grown on nutrient agar (0.1% Lab Lemco, 0.2% yeast extract, 0.5% bacteriological peptone, 0.5% NaCl, 1.5% agar) for 24 h at 28°C. Grown cultures were transferred on Trypticase Soy Agar (TSA) plates, containing 3% Trypticase Soy Broth (BBL) and 1.5% Bacto-Agar (Difco), for exactly 24 h at 28°C (+ 1°C). A loopful of cells was harvested with a sterile loop (diameter, 4 mm) and transferred to a test tube capped with a Teflon-lined screw-cap. The extraction and preparation of FAMES was performed as described by Stead (1989). FAME profiles were obtained by gas-liquid chromatography using a model 5980A gas chromatograph (HewlettPackard Co., Avondale, PA) equipped with a 5% phenyl methyl silicone capillary column (25 m x 0.2 mm; Hewlett-Packard), an automated sampler, a flame ionization detection system and an integrator as described by Korn-Wendish et al. (1989). FAME fingerprints were registered and treated by the Microbial Identification System software package (MIS version 3.7, MIDI, Microbial ID, Inc., Newark, DE) and a standardized calibration mixture. FAME profiles were compared by UPGMA analysis of Euclidean distance coefficient. 2.5. Calcite clearing test
The possible role of the bacterial isolates in stone degradation was examined by a zone-clearing test on a modified calcite-containing Deveze medium (NORMAL, 19881. A bottom layer of 30 ml Deveze medium (without calcite) was poured into Petri dishes (diameter 12 cm), on top of which 6 ml of separately sterilized Deveze medium containing 0.2% calcite was poured. Thirty strains were inoculated in duplicate, one plate incubated at 19°C the other at 25°C whereas all others were incubated at 25°C. Dissolution of the calcite was followed during 3 months. 2.6. Reference strains
Thirty-one reference strains were included in the study of which 24 were Micrococcus strains and the remainder were the type strains of nine Arthrobacter species (Table 1).
3. Results
167 (1995) 241-247
243
and discussion
The method described by Lewis and May (19851, using 0.001% Tween 80 as surfactant combined with vigorous agitation for 1 h was used to detach the microorganisms from the stone. PYGV as a low nutrient medium - total organic carbon (TO0 content of 0.044% without agar (Palmer et al., 1991) - for the enumeration of chemoorganatrophic bacteria was chosen to mimic the low nutrient levels in natural stones. Counts of lo”-10’ per gram of stone of heterotrophic bacteria were found (Table 2), similar to the heterotrophic bacterial counts reported by Tayler and May (19911, Warscheid et al. (1989), and Lyalikova and Petushkova (1991). The pH of the stone samples (Table 2) varies between 7.6 and 9.3. The differences in pH values can be explained by the fact that the main wind direction is south-west, resulting in an exposure to direct rainfall which washes off all acidic deposition. At the northern side, a slightly lower pH was observed. The pH of freshly quarried Ledestone is about pH 8.5. Stone samples from the northern and southern sides of the cathedral showed the highest bacterial counts, whereas the western side with the highest pH (pH 9.3) showed the lowest bacterial count. Only 22 from the 232 isolated bacterial strains were Gram-negative, mostly rods. The remaining 210 strains were all Gram-positive cocci. The dominant colony type was smooth and round, white to yellow pigmented. No spore formation was recorded. A problem occurred when we wanted to prepare the isolates for FAME analysis. From the 232 bacterial strains isolated on PYGV medium, only 162 strains (70%) grew on NA (first step in FAME analysis). Tayler and May (1991) already stressed the fact that reduced strength medium gave superior recovery, demonstrating that several bacterial isolates from stone do not grow on nutrient rich medium. PYGV medium 100 times diluted supported primarily fungal growth and no bacterial growth (Palmer, 19911, suggesting that the strength of PYGV medium is probably good, although an ideal medium should be composed of nutrients similar to those found in situ.
244
P. Descheemaeker,
J. Swings /Science
of the Total Environment
167 (1995)
241-247
Table 1 Reference strains incorporated in FAME analysis, the last column represents the identification similarity with the data base Name as received Micrococcus agilis kristinae luteus
lylae
LMGa No
No as receivedb
14213T 14215T 14216
CCM 2390 CCM 2690 CCM 2692 ATCC 4698 ATCC 10240 ATCC 9341 CCM 144 CCM 2693 ATCC 27569 ATCC 27567 CCM 2694
4050T 3293 8816
14217 14218’ 14192 14193
14219
nishinomiyaensis
14220 14221 14222T
roseus
14223 14224T 14225 14226 14221
sedentarius
1422ST 14229 14230
varians
14231T 14232
Arthrobacter artrocyaneus aurescens crystallopoietes globifonnis histidinolovorans ilicis oxydans polychromogenes ureafaciens
3814T
3815T 3819T 3813T 3822T 3659T 3816T
3821 3812T
CCM2695
CCM CCM CCM CCM CCM CCM CCM CCM CCM CCM CCM CCM
2696 2140 2669 679 560 839 633 314 2697 2699 884 541
CCM 1645 CCM 1649 CCM 2386 ATCC 8010 ATCC 11442 ATCC 14264 ATCC 14358 CBRI 21038 CCM 1644
Identification by FAME
At level
M. roseus
0.704
M. M. M. M. M. M. M. M. M. M. M. M.
kristinae kristinae luteus A lylae A luteus A luteus B lylae A lylae A !ylae B lylae B lylae A lylae A
0.914 0.838 0.233 0.300
0.914 0.705
0.551 0.435
0.701 0.568 0.842 0.375
N.M.C N.M. M. M. M. M. M. M. M. M. A.
roseus lylae roseus roseus sedentarius sedentatius sedentatius varians protophonniae
0.824 0.025 0.434 0.311 0.037 0.323 0.609 0.908 0.758
A. M. A. A. A. A. A. A. A.
mysorens luteus artrocyaneus globifotmis artrocyaneus ureafaciens oxydnns oxydans protophotmiae
0.693 0.463 0.428 0.712 0.652 0.676 0.517
0.195 0.845
aLMG, Laboratorium voor Microbiologic Gent Culture Collection, Universiteit Gent, Belgium. bCCM, Czechoslovak Collection of Microorganisms, Bmo, Czechoslovakia; ATCC, American Type Culture Collection, Rockville, U.S.A.; CBRI, Chemistry Research Institute, Research Branch, Department of Agriculture, Ottawa, Ontario, Canada. ‘N.M., No match with the MIS database.
The 24 Micrococcus reference strains (comprising eight of the nine described species (Kocur, 1986)) could be differentiated by FAME (Table 1). M. nishinomiyuensisis not included in the MIS database but formed a clearly distinct cluster. The strains M. agilis LMG 14213T and M. varians LMG 14232 gave aberrant identifications for which no plausible explanation can be given. M. luteus and M. lylae showed some overlap and it is
not clear if FAME analysis allows the separation of these closely related species. Low similarity values with the MIS database may be due to the limited number of Micrococcus entries in the commercial TSBA database (MIS). Both M. luteus and M. Jylae are divided into two subgroups A and B in the MIS database. M. lylae subgroup A and B together with 44. luteus subgroup A are related, whereas hf. luteus sub-
P. Descheemaeker,
J. Swings /Science
of the Total Environment
Table 2 Mean pH values and mean total heterotrophic bacterial counts (per gram stone) from the Lede-stone samples of the St. Bavo Cathedral in Ghent Side of the cathedral” North (5) South (7) West (2) 7.6 8.9 9.3 PH Heterotrophic bacterial count 1.1 x 10’ 9.7 X 10h 4.9 X lo4 “Number of samples in parenthesis.
group B seems not to be related to the former three subgroups. It is not yet clear if the currently recognized Micrococcus species represent ‘natural species’ or if they encompass all of the natural variation that exists within the genus (Alderson et al., 1991). The respective subgroups may represent this variation. Arthrobacter can only be separated at genus level by FAME analysis. A. aurescensLMG 3815* was identified as M. luteus at a level of 0.5. It is known that arthrobacters and micrococci are closely related and are phylogenetically inseparable (Alderson et al., 1991). From the 162 unknown bacterial isolates studied by FAME analysis (Table 3), 140 strains were Gram-positive and 22 Gram-negative; five strains were identified as Arthrobacter species at levels from 0.4 to 0.7; identification to species level was not possible. The two M. kristinae reference strains (LMG 14215* and 14216) clustered Table 3 Identification Gram
Gramnegatives
245
together with nine bacterial isolates at levels from 0.4 to 0.9. These strains were mainly found on the southern side of the cathedral. One large cluster was composed of 83 unknown bacterial isolates together with four M. 1yZaereference strains (LMG 14193,14219,14220, 14218T) and the type strain of J4. futeus LMG 4050*. All the strains in this cluster were identified, at low similarity levels, as M. fylae subgroup A and were equally distributed over the three sides of the cathedral. Three strains were identified as i’t4. luteus subgroup B at levels 0.5,0.6 and 0.7. Six other strains belong to M. Zuteussubgroup A at levels 0.7-0.9. It is possible that the well identified M. iuteus strains are from human origin whereas the Arthrobacter and M. Iylae strains are typical for the natural stone inhabiting flora. From the 22 Gram-negative strain, seven were related to Pseudomonasvesicularis. A number of Gram-negative and Gram-positive isolates formed separate groups which could not be identified. The possible role of the bacterial isolates in stone degradation was examined by a zone-clearing test on a modified calcite containing Deveze medium (NORMAL, 1988). To prevent drying out of the medium during prolonged incubation, a bottom layer of Deveze medium without calcite was poured into the Petri dishes and a top layer of calcite containing Deveze medium was poured onto this.
and distribution of the bacterial isolates Identification
Side of the cathedral North
Gram-positives
167 (1995) 241-247
Arthrobacter Micrococcus kristinae Micrococcus lylae Micrococcus luteus
Not identified Pseudomonas
sp.
1
South 4 8
1
33
41
9
8
I
17 7
13 -
14
1
vesicularis
Not identified
West
4
246
P. Descheemaeker,
J. Swings /Science
of the Total Environment
From 152 strains which were tested for their calcite dissolving capacity, only 29 strains revealed clearing zones. Six A4. luteus (subgroup A) strains together with three unidentified closely related Gram-positive strains showed clearing zones after 1 week of incubation at 2X, whereas the other strains showed clearing zones only after 14 days of incubation. Prolonged incubation up to 3 months did not reveal significant changes. To check the influence of temperature on the acid production, 30 strains were incubated at 19°C and at 25°C. No difference in the calcite dissolving capacity was detected. Only a small number of bacterial isolates was able to produce sufficient organic acids to dissolve CaCO,. In nature however, these bacteria live and survive under nutrient limitations and under unfavourable external conditions. Although we have no evidence at hand, we think that the acid production by these bacteria might be weak in situ. Consequently, biodeterioration by biochemical action of heterotrophic bacteria might be a negligible phenomenon. Nevertheless, it is very hard to imagine that the presence of large numbers of heterotrophic bacteria in and on natural building stones would have no effect at all on the stone whatsoever. In forming consortia with other microorganisms in biofilms, the heterotrophs might participate directly or indirectly to biodeterioration through physical mechanisms. Acknowledgements This work has been supported by the EC (project EV5V-CT92-0112). References Alderson, G., E.N. Amadi, G. Pulverer and S. Zai, 1991. Recent advances in the classification and identification of the genus Micrococcus. In: J. Jeljaszewicz and P. Ciborowski (Ed& The Staphylococci. Zbl. Bak. Suppl. 21. Fischer, Stuttgart, pp. 103-109. Bassi, M., A. Ferrari, M. Realini and C. Sorlini, 1986. Red stains on the Certosa of Pavia: a case of biodeterioration. Int. Biodeter., 22: 201-205. Eckhardt, F.E.W., 1985. Solubihzation, transport, and deposition of mineral cations by microorganisms - efficient rock weathering agents. In: J.I. Drever (Ed.), The Chemistry of Weathering. Reidel, Dordrecht, pp. 161-173.
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Kocur, M., 1986. Genus I. Micrococcus Cohn 1872, 151AL. In: P.H.A. Sneath, N.S. Mair, M.E. Sharpe and J.G. Holt (Eds.), Bergey’s Manual of Systematic Bacteriology, Vol. 2. Williams and Wilkins, Baltimore, pp. 1004-1008. Kom-Wendish, F., A. Kempf, E. Grund, R.M. Kroppenstedt and H.J. Kutzner, 1989. Transfer of Faenia rectivitgula Kurup and Agre 1983 to the genus Saccharopolyspora Lacey and Goodfellow 1975, elevation of Saccharopoiyspora hirsuta subsp. taberi Labeda 1987 to species level, and ammended description of the genus Saccharopolyspora. Int. J. Syst. Bacterial., 39: 430-441. Lewis, F. and E. May, 1985. Isolation and enumeration of autotrophic and heterotrophic bacteria from decayed stone. In: G. Felix (Ed.), Proceedings of the Vth International Congress on Deterioration and Conservation of Stone, Vol. 2. Presses Polytechniques Romandes, Lausanne, pp. 633-642. Lewis, F., E. May and B. Daley, 1988. The role of heterotrophic bacteria in the decay of sandstone from ancient monuments. In: L.H.G. Morton (Ed.), Biodeterioration of Constructional Materials. Biodeterioration Society, Kew, pp. 45-53. Leysen, L.A., E.J. Roekens and R.E. Van Grieken, 1990. Characterization of the weathering crust of various historical buildings in Belgium. Sci. Total Environ., 90: 117-147. Lyalikova, N.N. and Y.P. Petushkova, 1991. Role of microorganisms in the weathering of minerals in building stones of historical buildings. Geomicrobiol. J., 9: 91-101. May, E., F.J. Lewis, S. Pereira, S. Tayler, M.R.D. Seaward and D. Allsopp, 1993. Microbial deterioration of building stone - a review. Biodeter. Abstr., 7: 109-123. Nijs, R., 1985. Petrographical characterization of calcareous building stones in northern Belgium. In: G. Felix (Ed.), Proceedings of the Vth International Congress on Deterioration and Conservation of Stone, Vol. 1. Presses Polytechniques Romandes, Lausanne, pp. 13-21. NORMAL, Commissione Normativa Manufatti Lapidei, 1988. Microflora autotrofa ed eterotrofa: Tecniche di isolamento in coltura. CNR-ICR, Rome, Document0 Normal 9/88. Palmer, R.J., 1991. Microbial communities involved in the weathering of three historic buildings in northern Germany. In: N.S. Baer, C. Sabbioni and A.I. Sors (Eds.), Science, Technology and European Cultural Heritage. Butterworth Heinemann, Oxford, pp. 478-480. Palmer, R.J., 1992. Microbiological damage to building stone: analysis and intervention. In: R.G.M. Webster (Ed.), Stone Cleaning and the Nature, Soiling and Decay Mechanisms of Stone. Donhead Publishing, London, pp. 239-245. Palmer, R.J., J. Siebert and P. Hirsch, 1991. Biomass and organic acids in sandstone of a weathering building: production by bacterial and fungal isolates. Microbial Ecol., 21: 253-266. Rosvall, J., 1986. Air Pollution and Conservation: Safeguarding our Architectural Heritage. AB Volvo, Trycksaksservice, Gothenburg. Stead, D.E., 1989. Grouping of Xanthomonas campeshis
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pathovars of cereals and grasses by fatty acid profiling. EPPO Bull., 19: 57-68. Tayler, S. and E. May, 1991. The seasonal@ of heterotrophic bacteria on sandstones of ancient monuments. Int. Biodeter., 28: 49-64. Vreeland, R.H., 1991. The Family Halomonadaceae. In: A. Balows, H.G. Triiper, M. Dworkin, W. Harder and K.H. Schleifel (Eds.), The Prokatyotes. A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Application. Springer, New York, pp. 3181-3188. Warscheid, T., K. Petersen and W.E. Krumbein, 1988. Effect of cleaning on the distribution of microorganisms on rock
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247
surfaces. In: D.R. Houghton, R.N. Smith and H.O.W. Eggins (Eds.), Biodeterioration 7. Elsevier, New York, pp. 455-460. Warscheid, T., K. Petersen and W.E. Krumbein, 1989. Die Besiedlung unterschiedlicher Sandsteine durch chemoorganotrophe Bakterien und deren Einflul3 auf den ProzeB der Gesteinszerstorung. Z. dt. geol. Ges., 140: 209-217. Warscheid, T., M. Oelting and W.E. Krumbein, 1991. Physico-chemical aspects of biodeterioration processes on rocks with special regard to organic pollutants. Int. Biodeter., 28: 37-48.
The Science
of the Total
Environment
167 (1995)
241-247
The application of fatty acid methyl ester analysis (FAME) for the identification of heterotrophic bacteria present in decaying Lede-stone of the St. Bavo Cathedral in Ghent P. Descheemaeker*, J. Swings Laboratorium
voor Microbiologic,
Vakgroep
Biochemie, Fysiologie en Microbiologic, 35, B-9000 Ghent, Belgium
Universiteit
Gent, K. L. Ledeganckstraat
Abstract
The heterotrophic bacterial community presentin decaying Lede-sandstoneof the Cathedral of Ghent (Belgium) wasstudied.Two-hundred thirty-two heterotrophic bacterial strainswere isolatedof which 162were studied by fatty acid methyl ester analysis(FAME). One-hundred forty isolateswere Gram-positive; 101strainsbelong to the genus Micrococcus; nine strainswere identified as M. kristinae. One large cluster of 83 strainswasrelated to M. luteus/M. lylae. Other Gram-positive strainswere related to Arthrobacter. Of the 22 Gram-negativestrains,sevenstrainswere related to Pseudomonas uesiculati. A number of Gram-negativeand Gram-positive isolatesformed separateclusters which could not be identified. The possiblerole of the bacterial isolatesin stone degradationwas examinedby a zone-clearing test on a calcite-containingmedium. Of the 152 strains examined,only nine showedclearing zones after 7 days of incubation at 25°C 20 odd strainsshoweda dissolutionof calcite after 14 days, whereasall other strainsshowedno clearing zones,even after prolonged incubation (up to 3 months).The zone-clearingtest showed the samereactionswhen incubated at 24°C or at 19°C. Keywords: Fatty acid methyl ester analysis(FAME); Heterotrophic bacteria; St. Bavo Cathedral, Ghent; Micrococcus; Arthrobacter; Stone decay
1. Introduction Chemoorganotrophic bacterial growth on stone is dependent on the available carbon compounds for an energy and carbon source. Stonework seems to offer enough organic substances for heterotrophic growth (Rosvall, 1986). The organic
*Corresponding
author.
0048-9697/95/$09.50 0 1995 Elsevier SSDI 0048-9697(95)04585-B
Science
BV. All rights
reserved.
compounds may have different origins: (1) phototrophic organisms(cyanobacteria, algae, lichens) absorb sunlight as an energy source and CO, as a carbon source to establish their life cycle. Carbohydrates excreted by these organisms or the organic matter released after cell death can be used as carbon source for chemoorganotrophic growth; (2) a range of hydrocarbons originate from industrial, traffic or domestic burning pollu-
242
P. Descheemaeker, J. Swings
/Science
tants, dirt and dust particles (Warscheid et al., 1991) and (3) sedimentary rocks may contain diagenetic organic material sufficient to support chemoorganotrophic growth (Eckhardt, 198.5). Chemoorganotrophic bacteria are thought to play a role in stone decay (May et al., 19931, although experimental proof of microbial weathering under natural (low nutrient) conditions is still lacking. The mere occurrence of microorganisms on stone surfaces does not automatically imply destructive action, but these microbial populations may be involved in the aesthetic detrimental appearance of historic monuments (Warscheid et al., 1988). The involvement of microbial weathering processes on a specific building can only be assessed by a complete description of the microbial community and proof that this community is mechanistically responsible for deterioration (Palmer, 1992). Studies on the identification of the heterotrophic microbial populations present on decaying stone are rare. Predominant Gram-positive (Micrococcus) and Gram -negative (Flavobactetium, Pseudomonas) bacteria have been identified (Bassi et al., 1986; Lewis et al., 1988). Tayler and May (1991) found during the winter and spring months both Gram-negative (e.g. Flavobacterium / Cytophaga, Actinobacter) and Gram-positive (e.g. Corynebactetiurn, Micro-
coccus) bacteria whereas in the drier summer months, only Gram-positive forms (predominantly Bacillus sp.> were found, suggesting a seasonal variation. It was the objective of this study to identify the heterotrophic microbial population present on deteriorating Lede-stone of the St. Bavo cathedral in Ghent, and to determine their distribution over the different sampling sites. 2. Materials and methods 2.1. Description of the Lede-stone and study site
The deteriorating Lede-stone of the St. Bavo Cathedral of Ghent (Belgium) was sampled. The cathedral is largely built of Balegem (or Lede) stone. The Balegem stone is a light, yellowishgreyish, sandy limestone of Tertiary age. It contains a significant amount of rather fine detrital
of
the Total Environment 167 (1995) 241-247
quartz grains, and some glauconite and calcareous skeletons, cemented by a micro-crystalline calcium carbonate matrix. The total calcite content is 40-60%. It is the typical building material for the Gothic, Renaissance and Baroque monuments of Flanders (Nijs, 1985; Leysen et al., 1990). 2.2. Sample collection and treatment
Stone samples of lo-15 g were peeled off from the surface of severely deteriorated Lede-stones from the northern (five samples), the southern (seven samples) and the western side (two samples) of the cathedral, 1.5-2 m above the ground. Immediately after collection, the stone samples were crushed to a fine powder in an alcoholflamed mortar under a laminar flow hood. One gram of the powder was shaken for 1 h in 10 ml of a sterile water solution containing 0.001% Tween 80 and 0.85% NaCl (Lewis and May, 19851, diluted 100 times and spiral plated in triplicate on Petri dishes containing 30 ml PYGV medium to enumerate heterotrophic bacteria. The PYGV medium contains per litre: 0.025% bacteriological peptone, 0.025% yeast extract, 0.025% glucose, 20 ml Hutners mineral salts (Vreeland, 1991), 0.005% actidione and 1.5% agar. After sterilization, 10 ml of a vitamin solution was added containing per litre MilliQ water: 4 mg biotin, 20 mg pyridoxine hydrochloride, 10 mg thiamine hydrochloride, 10 mg Ca-pantothenate, 10 mg p-aminobenzoic acid, 4 mg folic acid, 10 mg riboflavin, 10 mg nicotinic acid and 0.2 mg vitamin B,z (Palmer et al., 1991). The pH of the stone samples was measured by suspending 1 g of the powdered stone in 15 ml of 1 M KCl. 2.3. Enumeration and isolation of chemoo%anotrophic bacteria
The inoculated PYGV media were incubated for 15 days at 25°C and counted each 5 days. Two-hundred thirty-two heterotrophic bacterial strains were isolated representative of the different dominant colony types. Each strain was purified on PYGV medium. Pure cultures were stored under liquid nitrogen. For each pure culture colony morphology, cell morphology, Gram stain, pigmentation and spore formation was recorded.
P. Descheemaeker,
2.4. Fatty acid methyl ester (FAME)
J. Swings /Science
of the Total Environment
analysis
All pure cultures were grown on nutrient agar (0.1% Lab Lemco, 0.2% yeast extract, 0.5% bacteriological peptone, 0.5% NaCl, 1.5% agar) for 24 h at 28°C. Grown cultures were transferred on Trypticase Soy Agar (TSA) plates, containing 3% Trypticase Soy Broth (BBL) and 1.5% Bacto-Agar (Difco), for exactly 24 h at 28°C (+ 1°C). A loopful of cells was harvested with a sterile loop (diameter, 4 mm) and transferred to a test tube capped with a Teflon-lined screw-cap. The extraction and preparation of FAMES was performed as described by Stead (1989). FAME profiles were obtained by gas-liquid chromatography using a model 5980A gas chromatograph (HewlettPackard Co., Avondale, PA) equipped with a 5% phenyl methyl silicone capillary column (25 m x 0.2 mm; Hewlett-Packard), an automated sampler, a flame ionization detection system and an integrator as described by Korn-Wendish et al. (1989). FAME fingerprints were registered and treated by the Microbial Identification System software package (MIS version 3.7, MIDI, Microbial ID, Inc., Newark, DE) and a standardized calibration mixture. FAME profiles were compared by UPGMA analysis of Euclidean distance coefficient. 2.5. Calcite clearing test
The possible role of the bacterial isolates in stone degradation was examined by a zone-clearing test on a modified calcite-containing Deveze medium (NORMAL, 19881. A bottom layer of 30 ml Deveze medium (without calcite) was poured into Petri dishes (diameter 12 cm), on top of which 6 ml of separately sterilized Deveze medium containing 0.2% calcite was poured. Thirty strains were inoculated in duplicate, one plate incubated at 19°C the other at 25°C whereas all others were incubated at 25°C. Dissolution of the calcite was followed during 3 months. 2.6. Reference strains
Thirty-one reference strains were included in the study of which 24 were Micrococcus strains and the remainder were the type strains of nine Arthrobacter species (Table 1).
3. Results
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and discussion
The method described by Lewis and May (19851, using 0.001% Tween 80 as surfactant combined with vigorous agitation for 1 h was used to detach the microorganisms from the stone. PYGV as a low nutrient medium - total organic carbon (TO0 content of 0.044% without agar (Palmer et al., 1991) - for the enumeration of chemoorganatrophic bacteria was chosen to mimic the low nutrient levels in natural stones. Counts of lo”-10’ per gram of stone of heterotrophic bacteria were found (Table 2), similar to the heterotrophic bacterial counts reported by Tayler and May (19911, Warscheid et al. (1989), and Lyalikova and Petushkova (1991). The pH of the stone samples (Table 2) varies between 7.6 and 9.3. The differences in pH values can be explained by the fact that the main wind direction is south-west, resulting in an exposure to direct rainfall which washes off all acidic deposition. At the northern side, a slightly lower pH was observed. The pH of freshly quarried Ledestone is about pH 8.5. Stone samples from the northern and southern sides of the cathedral showed the highest bacterial counts, whereas the western side with the highest pH (pH 9.3) showed the lowest bacterial count. Only 22 from the 232 isolated bacterial strains were Gram-negative, mostly rods. The remaining 210 strains were all Gram-positive cocci. The dominant colony type was smooth and round, white to yellow pigmented. No spore formation was recorded. A problem occurred when we wanted to prepare the isolates for FAME analysis. From the 232 bacterial strains isolated on PYGV medium, only 162 strains (70%) grew on NA (first step in FAME analysis). Tayler and May (1991) already stressed the fact that reduced strength medium gave superior recovery, demonstrating that several bacterial isolates from stone do not grow on nutrient rich medium. PYGV medium 100 times diluted supported primarily fungal growth and no bacterial growth (Palmer, 19911, suggesting that the strength of PYGV medium is probably good, although an ideal medium should be composed of nutrients similar to those found in situ.
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Table 1 Reference strains incorporated in FAME analysis, the last column represents the identification similarity with the data base Name as received Micrococcus agilis kristinae luteus
lylae
LMGa No
No as receivedb
14213T 14215T 14216
CCM 2390 CCM 2690 CCM 2692 ATCC 4698 ATCC 10240 ATCC 9341 CCM 144 CCM 2693 ATCC 27569 ATCC 27567 CCM 2694
4050T 3293 8816
14217 14218’ 14192 14193
14219
nishinomiyaensis
14220 14221 14222T
roseus
14223 14224T 14225 14226 14221
sedentarius
1422ST 14229 14230
varians
14231T 14232
Arthrobacter artrocyaneus aurescens crystallopoietes globifonnis histidinolovorans ilicis oxydans polychromogenes ureafaciens
3814T
3815T 3819T 3813T 3822T 3659T 3816T
3821 3812T
CCM2695
CCM CCM CCM CCM CCM CCM CCM CCM CCM CCM CCM CCM
2696 2140 2669 679 560 839 633 314 2697 2699 884 541
CCM 1645 CCM 1649 CCM 2386 ATCC 8010 ATCC 11442 ATCC 14264 ATCC 14358 CBRI 21038 CCM 1644
Identification by FAME
At level
M. roseus
0.704
M. M. M. M. M. M. M. M. M. M. M. M.
kristinae kristinae luteus A lylae A luteus A luteus B lylae A lylae A !ylae B lylae B lylae A lylae A
0.914 0.838 0.233 0.300
0.914 0.705
0.551 0.435
0.701 0.568 0.842 0.375
N.M.C N.M. M. M. M. M. M. M. M. M. A.
roseus lylae roseus roseus sedentarius sedentatius sedentatius varians protophonniae
0.824 0.025 0.434 0.311 0.037 0.323 0.609 0.908 0.758
A. M. A. A. A. A. A. A. A.
mysorens luteus artrocyaneus globifotmis artrocyaneus ureafaciens oxydnns oxydans protophotmiae
0.693 0.463 0.428 0.712 0.652 0.676 0.517
0.195 0.845
aLMG, Laboratorium voor Microbiologic Gent Culture Collection, Universiteit Gent, Belgium. bCCM, Czechoslovak Collection of Microorganisms, Bmo, Czechoslovakia; ATCC, American Type Culture Collection, Rockville, U.S.A.; CBRI, Chemistry Research Institute, Research Branch, Department of Agriculture, Ottawa, Ontario, Canada. ‘N.M., No match with the MIS database.
The 24 Micrococcus reference strains (comprising eight of the nine described species (Kocur, 1986)) could be differentiated by FAME (Table 1). M. nishinomiyuensisis not included in the MIS database but formed a clearly distinct cluster. The strains M. agilis LMG 14213T and M. varians LMG 14232 gave aberrant identifications for which no plausible explanation can be given. M. luteus and M. lylae showed some overlap and it is
not clear if FAME analysis allows the separation of these closely related species. Low similarity values with the MIS database may be due to the limited number of Micrococcus entries in the commercial TSBA database (MIS). Both M. luteus and M. Jylae are divided into two subgroups A and B in the MIS database. M. lylae subgroup A and B together with 44. luteus subgroup A are related, whereas hf. luteus sub-
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Table 2 Mean pH values and mean total heterotrophic bacterial counts (per gram stone) from the Lede-stone samples of the St. Bavo Cathedral in Ghent Side of the cathedral” North (5) South (7) West (2) 7.6 8.9 9.3 PH Heterotrophic bacterial count 1.1 x 10’ 9.7 X 10h 4.9 X lo4 “Number of samples in parenthesis.
group B seems not to be related to the former three subgroups. It is not yet clear if the currently recognized Micrococcus species represent ‘natural species’ or if they encompass all of the natural variation that exists within the genus (Alderson et al., 1991). The respective subgroups may represent this variation. Arthrobacter can only be separated at genus level by FAME analysis. A. aurescensLMG 3815* was identified as M. luteus at a level of 0.5. It is known that arthrobacters and micrococci are closely related and are phylogenetically inseparable (Alderson et al., 1991). From the 162 unknown bacterial isolates studied by FAME analysis (Table 3), 140 strains were Gram-positive and 22 Gram-negative; five strains were identified as Arthrobacter species at levels from 0.4 to 0.7; identification to species level was not possible. The two M. kristinae reference strains (LMG 14215* and 14216) clustered Table 3 Identification Gram
Gramnegatives
245
together with nine bacterial isolates at levels from 0.4 to 0.9. These strains were mainly found on the southern side of the cathedral. One large cluster was composed of 83 unknown bacterial isolates together with four M. 1yZaereference strains (LMG 14193,14219,14220, 14218T) and the type strain of J4. futeus LMG 4050*. All the strains in this cluster were identified, at low similarity levels, as M. fylae subgroup A and were equally distributed over the three sides of the cathedral. Three strains were identified as i’t4. luteus subgroup B at levels 0.5,0.6 and 0.7. Six other strains belong to M. Zuteussubgroup A at levels 0.7-0.9. It is possible that the well identified M. iuteus strains are from human origin whereas the Arthrobacter and M. Iylae strains are typical for the natural stone inhabiting flora. From the 22 Gram-negative strain, seven were related to Pseudomonasvesicularis. A number of Gram-negative and Gram-positive isolates formed separate groups which could not be identified. The possible role of the bacterial isolates in stone degradation was examined by a zone-clearing test on a modified calcite containing Deveze medium (NORMAL, 1988). To prevent drying out of the medium during prolonged incubation, a bottom layer of Deveze medium without calcite was poured into the Petri dishes and a top layer of calcite containing Deveze medium was poured onto this.
and distribution of the bacterial isolates Identification
Side of the cathedral North
Gram-positives
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Arthrobacter Micrococcus kristinae Micrococcus lylae Micrococcus luteus
Not identified Pseudomonas
sp.
1
South 4 8
1
33
41
9
8
I
17 7
13 -
14
1
vesicularis
Not identified
West
4
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From 152 strains which were tested for their calcite dissolving capacity, only 29 strains revealed clearing zones. Six A4. luteus (subgroup A) strains together with three unidentified closely related Gram-positive strains showed clearing zones after 1 week of incubation at 2X, whereas the other strains showed clearing zones only after 14 days of incubation. Prolonged incubation up to 3 months did not reveal significant changes. To check the influence of temperature on the acid production, 30 strains were incubated at 19°C and at 25°C. No difference in the calcite dissolving capacity was detected. Only a small number of bacterial isolates was able to produce sufficient organic acids to dissolve CaCO,. In nature however, these bacteria live and survive under nutrient limitations and under unfavourable external conditions. Although we have no evidence at hand, we think that the acid production by these bacteria might be weak in situ. Consequently, biodeterioration by biochemical action of heterotrophic bacteria might be a negligible phenomenon. Nevertheless, it is very hard to imagine that the presence of large numbers of heterotrophic bacteria in and on natural building stones would have no effect at all on the stone whatsoever. In forming consortia with other microorganisms in biofilms, the heterotrophs might participate directly or indirectly to biodeterioration through physical mechanisms. Acknowledgements This work has been supported by the EC (project EV5V-CT92-0112). References Alderson, G., E.N. Amadi, G. Pulverer and S. Zai, 1991. Recent advances in the classification and identification of the genus Micrococcus. In: J. Jeljaszewicz and P. Ciborowski (Ed& The Staphylococci. Zbl. Bak. Suppl. 21. Fischer, Stuttgart, pp. 103-109. Bassi, M., A. Ferrari, M. Realini and C. Sorlini, 1986. Red stains on the Certosa of Pavia: a case of biodeterioration. Int. Biodeter., 22: 201-205. Eckhardt, F.E.W., 1985. Solubihzation, transport, and deposition of mineral cations by microorganisms - efficient rock weathering agents. In: J.I. Drever (Ed.), The Chemistry of Weathering. Reidel, Dordrecht, pp. 161-173.
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pathovars of cereals and grasses by fatty acid profiling. EPPO Bull., 19: 57-68. Tayler, S. and E. May, 1991. The seasonal@ of heterotrophic bacteria on sandstones of ancient monuments. Int. Biodeter., 28: 49-64. Vreeland, R.H., 1991. The Family Halomonadaceae. In: A. Balows, H.G. Triiper, M. Dworkin, W. Harder and K.H. Schleifel (Eds.), The Prokatyotes. A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Application. Springer, New York, pp. 3181-3188. Warscheid, T., K. Petersen and W.E. Krumbein, 1988. Effect of cleaning on the distribution of microorganisms on rock
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